Celiac disease is an autoimmune digestive disorder in which intestinal lesions develop in genetically susceptible individuals in response to the ingestion of specific cereal seed storage proteins. The only treatment is strict adherence to a diet free from the offending grains. Therefore, considerable research has been devoted to identifying the specific amino acid sequences in wheat seed storage proteins responsible for eliciting the immune reaction in celiac patients, and this research has motivated the hope that strains of wheat and other grains could be developed in which these proteins have been reduced or eliminated. During the course of NIH STTR phase I research conducted in collaboration between Dr. Diter von Wettstein of Washington State University and Arcadia Biosciences, we have cloned and begun a systematic analysis of the composition of the previously uncharacterized ? gliadin gene family located on homoeologous chromosome 6D of hexaploid wheat. Among this gliadin gene family there exist one or more genes for the gliadin subtype immunodominant in celiac disease containing a 33-mer peptide highly resistant to digestion (Molberg et al., Gastroenterology 128:393-401,2005;Shan et al., Science 297:2275-2279,2002). Identifying the gene(s) encoding this gliadin subtype is a prerequisite for using TILLING to attempt to eliminate it from the wheat grain. TILLING (Targeting Induced Local Lesions in Genomes) is a novel, non-GMO (genetically modified organism) technique invented by an Arcadia Biosciences scientist that is a target-selected variation of mutation breeding. During STTR phase I research we have also determined the importance of a DNA 5- methylcytosine deglycosylase for gluten gene expression in wheat and barley and are targeting the wheat homologues for inactivation via TILLING and transgenic means. Finally, we have developed a large number of novel wheat lines containing missense and nonsense mutations in candidate transcription factors known to be important for the accumulation of seed storage proteins toxic to celiac patients. STTR phase II research will continue progress towards the development of wheat acceptable for celiac patients by: 1. Using TILLING and transgenic approaches to develop wheat plants down-regulated for endosperm DNA 5- methylcytosine deglycosylation activity and characterizing their seed storage composition. 2. Making crosses between plants harboring priority missense and nonsense alleles in the candidate transcription factors and characterizing the phenotypes of the progeny. The outcome of this NIH STTR Phase II research will be mutant and transgenic wheat lines whose creation are a necessary prerequisite for the commercialization of wheat cultivars acceptable for sufferers of celiac disease. PUBLIC HEALTH RELEVANCE: The proposed research takes an innovative approach to ameliorating the cause of celiac disease by developing wheat varieties that lack the major gluten components that elicit the disease. Celiac disease is the most common food-sensitive enteropathological condition in humans with a prevalence of 0.5-1%, and currently the only treatment for patients is the complete exclusion of cereal proteins from the diet. Developing wheat that is acceptable for celiac patients thus addresses an urgent, unmet need.